Insight into the hetero-interactions of 4-nonylphenol with dissolved organic matter: multiple spectroscopic methods, 1H NMR study and principal component analysis

Understanding the interactions between heterogeneous dissolved organic matter (DOM) and nonylphenols (NPs) is essential for predicting their behavior and fate in the environment. Herein, we firstly obtained different MW-fractionated humic acids (HAs) using the ultrafiltration method. Afterward, the molecular weight (MW)-dependent interactions of HAs with 4-nonylphenol (4-NP) were analysed by excitation emission matrix (EEM) fluorescence spectroscopy, fluorescence quenching, UV-vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA). EEM spectra indicated that the quenching mechanism was static. In the binding process, the higher MW fractions showed stronger interaction with 4-NP than the lower MW counterparts, exhibiting a clear MW-dependent interaction heterogeneity. The interaction constants for the 4-NP–HAs system were suppressed as the ionic strength decreased and pH increased, which was especially obvious in the binding of 4-NP to the lower MW-fractionated HAs. The FTIR spectra revealed that hydroxyl and aromatics were involved in the interaction process of HA fractions with 4-NP. It was also found from 1H NMR that π–π interactions between aromatic rings of 4-NP and MW-fractionated HAs were responsible for the complexation. The correlation analysis and PCA results indicated that aromaticity and MW play important roles in the interaction process and confirmed an obvious interaction heterogeneity among MW-fractionated HAs samples. This work highlighted MW-dependent interaction heterogeneities of HA, which suggested that heterogeneity in MW distribution should be taken into consideration when exploring the fate and biogeochemistry cycling of 4-NP from contaminated environments.


Introduction
Nonylphenol ethoxylates (NPEOs), as one of the crucial classes of nonionic surfactants, are widely used in agricultural, domestic, and industrial elds. 1 As a result of chemical and biological processes, NPEOs can be degraded stepwise by losing ethoxylates to form more toxic nonylphenols (NPs). 2 Nowadays, NPs have been broadly detected in various environmental matrices, including water, soil, and atmosphere. 1,3-5 They were found to mimic the natural hormone oestrogen, resulting in disrupting the normal functioning of the hormonal system. Due to these disturbing results, NPs were designated as priority hazardous substances in European Union. 6 Currently, investigating the occurrence, fate, and potential ecological risks of NPs in the aquatic, terrestrial, and atmospheric environments is urgent. 1,[3][4][5] Due to the nonyl chain, NPs have hydrophobic properties. 4 The highly hydrophobic NPs pose a strong intendancy to interaction with soil/sediment organic matter, especially the ubiquitous dissolved organic matter (DOM), which signicantly inuence the transportation, transformation, and bioavailability of NPs in the environments. 7-10 By using dialysis equilibrium experiments, Li et al. 10 found DOM had high affinity to adsorb NPs. Liao et al. 8 investigated the kinetics and thermodynamics of NP sorption to the DOM, and the results indicated that the pseudo rst order model and Freundlich model could better describe the sorption process. Because of the interactions between DOM and NPs, the adsorption of NPs was inhibited by magnetic reduced graphene oxides. Unfortunately, DOM is a well-known heterogeneous mixture in the environment. However, till now, the previous works about 4-NP-HA interaction mainly focused on the bulk HA samples, ignoring the effects of the heterogeneities within the bulk HA on the environmental behaviors.
DOM is a complex and heterogeneous mixture of organic macromolecules consisting of different sizes, structures, and chemical properties. 11,12 The interactions between pollutants with DOM molecules were signicantly related to the organic compound species and MW distribution. For example, some studies revealed that high molecular weight MW-fractionated HAs which exhibited higher aromaticity and hydrophobicity had superior sorption ability on the other hydrophobic organic compounds (HOCs). [12][13][14][15] Nevertheless, recent researches observed that lower molecular weight MW-fractionated DOM (<1 kDa) had strong adsorption affinities on bisphenol A, octylphenol and pentachlorobenzene. 16,17 This discrepancy may be attributed to the composition heterogeneity of DOM and non-uniformity of HOCs binding functionalities in DOM. Therefore, understanding the interactions between different MW DOM and NPs under different environmental conditions, particularly the underlying interaction mechanisms, is imperative.
The interaction of HOCs with DOM has been investigated using various techniques, such as dialysis equilibrium, passive dosing, solubility enhancement, and uorescence quenching. [18][19][20][21] Among them, uorescence quenching is a popular and powerful method due to its sensitivity, non-destructivity, and simplicity. 22 It has been reported as an efficient method to investigate the interaction mechanism between HOCs and uorescent substances. 11,16,20 However, the binding sites are easily overlooked when exploring interactions. 20 Nuclear magnetic resonance (NMR) can be used to provide specic, molecular-level information involved in the interaction, and it is also applied to investigate those occurring between environmental pollutants and DOM. 23,24 Additionally, principal component analysis (PCA) is a multivariate statistical technique that can obtain the main comprehensive inuencing factors of interactions. However, limited information has been probed into the interaction mechanism between DOM and multiantibiotic situations. Therefore, combining these techniques can better understand the interaction mechanism between DOM and NPs deeply regarding binding capacities and binding sites.
The aim of this paper was to provide insights into the heterointeractions of NP with DOM in the natural environment. To achieve this purpose, 4-nonylphenol (4-NP) and a soil HA, were selected as the representative NP and DOM, respectively. We utilized the ultraltration technique was applied to fractionate the HA samples into different MW-fractionated HAs. Furthermore, the interaction properties between MW-fractionated HAs and 4-NP were investigated at different temperatures/pHs/ionic strengths by uorescence quenching method. UV-visible spectroscopy and FTIR spectroscopy were introduced to further explore the internal mechanism of the interaction of 4-NP with MW-fractionated HAs. The possible interaction sites were discussed by 1 H NMR analysis. The correlation analysis and principal component analysis were performed to understand the hetero-interaction. The results provide new insights into the binding interactions of the MW-dependent interaction heterogeneities of HA with 4-NP, which can improve our understanding of the fate and bioavailability of HOCs in aquatic environments.

Materials
4-NP (>99.5%), NaOD, DMSO-D 6 and D 2 O were purchased from Sigma-Aldrich. Physicochemical properties of 4-NP were listed in Table S1. † According to the method of the International Humic Substances Society (IHSS), HA was extracted from soil samples, collected from an undisturbed area at Dunhua, Jilin Province, China (43 18 0 36 00 N, 128 23 0 26 00 E). Amicon Ultra-15 centrifugal lters with nominal molecular weight cut-off of 3 kDa, 10 kDa, 30 kDa, and 100 kDa were obtained from Millipore (Darmstadt, Germany). The other reagents were purchased from Guangzhou Chemical Reagent (Guangzhou, China). If not specied, all the reagents were used as received without further purication. Ultrapure water (18.2 U cm À1 , purication by a Millipore system) was used throughout the experiments.

Fractionation and characterization of HA
The stock solution of HA was stirred overnight, followed by ltering through a 0.22 mm cellulose acetate lter paper (Millipore, Billerica, MA, USA). The ltrate was referred to as pristine HA. The HA fractions were obtained by using the Amicon Ultra-15 centrifugal lters and the detailed fractionation procedures were given elsewhere. 25 The fractions achieved were named >100 kDa HA, 30-100 kDa HA, 10-30 kDa HA, 3-10 kDa HA, and <3 kDa HA. Concentrations of pristine and MW-fraction HAs were quantied using a total organic carbon (TOC) analyzer (TOC-VCPH, Shimadzu, Japan). The pristine-and MWfractionated HAs were stored in the dark at 4 C until use. The MW distribution, elemental compositions, UV-visible absorption spectra, size exclusion chromatography (SEC), and excitation-emission matrices (EEM) uorescence spectra of pristine-and MW-fractionated HAs were characterized and the results were are given in the ESI. †

Fluorescence quenching experiments
The interaction properties of pristine and MW-fractionated HAs with 4-NP were examined by using the uorescence quenching method. Batch interaction experiments were conducted using several glass vials lled with MW-fractionated HA, then the 4-NP stock solution was spiked into the HA solutions. The corresponding electrolyte solution was added to each vial to maintain a nal volume of 10 mL, HAs at 0.5-10 mg C L À1 and 4-NP at 1 mg L À1 . The pH was adjusted using HNO 3 or NaOH. The IS was modied by adding NaNO 3 . Aer mixing, the vials were gently shaken by an oscillator for 4 h at 288 K, 298 K, and 308 K. This time period was adequate for reaction equilibrium based on preliminary experiments. Aer the equilibrium period, UVvisible absorbance spectra and EEM uorescence spectra were measured.

Spectral measurement
The UV-visible absorbance spectra were measured between 200 and 800 nm using a spectrophotometer (UV-2550, Shimadzu, Japan) with 1 cm quartz cells. If not specied, the concentration of HAs was 5 mg C L À1 . To get better FTIR spectra of 4-NP, HAs, and their complexes, the high concentration solutions of samples were prepared and freeze-dried. And all the obtained particles were homogenized and pressed under the irradiation of an infrared lamp to eliminate the effect of moisture in the samples. The FTIR spectra were recorded in a PerkinElmer frontier spectrometer equipped with a Germanium attenuated total reection accessory and a KBr beam splitter with 32 scans at a resolution of 4 cm À1 . All 1 H NMR spectra data were collected on a Bruker Avance 400 MHz instrument at 400. 13 MHz with a 5 mm Bruker inverse broadband probe. The temperature was set as 298 AE 1 K. Typically, 1 mg 4-NP was rstly added with 50 mL DMSO-D 6 to ensure 4-NP dissolution, and then diluted in 0.1 mol L À1 NaOD solution with the nal concentration of 120 mg mL À1 , and then the solid pristine and MW-fractionated HAs were added, respectively. The concentration ratio of HA with 4-NP in all samples was 0.5. The alkaline conditions were favorable for the dissolution of 4-NP and HAs. The resulting solution was sonicated for 120 s and transferred to NMR tubes. High concentrations of HAs and 4-NP were used to enhance the intensity of 1 H NMR and thus improve the signal-to-noise ratio of the analysis. 1 H NMR spectra were referenced to the chemical shi of the solvent being 4.709 ppm. All spectra were processed by MestReNova soware. All samples were performed in triplicate and the results in average were reported.

Data analysis
All uorescence spectra were corrected for the inner lter effect as follows: 26 where F obs and F were the uorescence intensities before and aer correction, and A ex and A em were the absorbance of the test solution at excitation and emission wavelengths, respectively. The interaction parameters of 4-NP to the pristine and MWfractionated HAs were calculated using the Stern-Volmer equation: 20 where F 0 and F were the corrected uorescence intensities of 4-NP without and with pristine and MW-fractionated HAs. k q was the energy transfer rate (L mol À1 s À1 ), s 0 was the lifetime of uorescence (3.63 ns). 27 [HA] was the added concentration of HA and K HA was the sorption coefficient. Because the HA fraction possessed intrinsic uorescence at excitation wavelength of 4-NP. In order to remove background effects, a blank was made for each concentration of HA fraction. The blank spectrum was subtracted from the emission spectrum of the corresponding 4-NP-HA solution. 20 In addition, the interaction constant (K) and the number of interaction sites (n) according to uorescence titration experiments were calculated from the site-binding equation for an interaction process involving a static quenching mechanism: 28 where F 0 and F were the corrected uorescence intensities of 4-NP in the absence and presence of pristine and MWfractionated HAs.
[HA] was the added concentration of HA (mg C L À1 ). The thermodynamic parameters for uorescence quenching of 4-NP by HAs, including the enthalpy change (DH), the entropy change (DS), and the Gibbs free energy change (DG), were used to interpret the interaction forces. The values of DH and DS can be estimated from eqn (4): where R was the ideal gas constant (8.314 J mol À1 K À1 ), T was the absolute temperature and K was the interaction constant at temperature T. Then the DG could be determined using the following eq: Details of the data analysis for the interaction of pristine and MW-fractionated HAs with 4-NP were given in the ESI. †

Physicochemical properties of the pristine and MWfractionated HAs
Separated by ultraltration, the MW-fractionated HAs showed apparent increases in molecular weights ranging from 2.30 to 20.9 kDa (Fig. S1 and Table S2 †). Elemental compositions and atomic ratios of the pristine and MW-fractionated HAs were listed in Table S2. † With the enhancement of molecular weights of the MW-fractionated HAs, an increasing and a decreasing trend was observed for the H/C ratios and the O/C ratios, respectively, indicated that the high molecular weight MWfractionated HAs contained more unsaturated hydrocarbons and less oxygen-containing functional groups. [29][30][31] Fig. 1a shows that UV-visible absorbance of the pristine and MW-fractionated HAs decreased monotonously as the wavelength increased and no absorption peak was observed. Except for 30-100 kDa HA, SUVA 254 values of molecular weight MW-fractionated HAs which were calculated via the division of the absorbance at 254 nm with TOC concentration, decreased from 9.24 to 6.99 with decreasing molecular weight of the MW-fractionated HAs. Polarity which dened as the elemental ratio of (O + N)/C increased from 0.51 for >100 kDa HA to 0.65 for <3 kDa HA. These results indicated that the higher molecular weight MWfractionated HAs (>30 kDa HA) contained more aromatic compounds and hydrophobic structures. 29,32,33 As presented in Fig. 1b, with decreasing molecular weight of the MW-fractionated HA, the absorbances of peaks at 3405, 1714 and 1240 cm À1 were increased slightly, corresponding to the O-H stretching of carboxyl acids, phenols and alcohol, C]O stretching of carboxylic acid, aldehyde and ketone, and C-O stretching of ester, ethers and phenols, respectively, 30 suggesting that the lower molecular weight MW-fractionated HA contained more carboxyl groups, consistent with the elemental analysis.
One uorescence peak (Ex/Em, Ex: 280-305 nm; Em: 435-474 nm) in each sample was observed (Fig. 2), indicating that the uorophore was the humic acid-like structure. 21 Generally, the greater abundance of aromatic chromophore possessed, the more red-shi of Em maxima was observed. 29,34 As the molecular weight of the MW-fractionated HAs increased, the peak Em wavelengths shied towards the longer wavelengths, which suggested more condensed and polymerized structures in higher molecular weight counterparts. Furthermore, the uorescence intensity of HA was positively related with the content of electron donating groups, such as hydroxyl and methoxyl groups. [35][36][37] These groups could improve the uorescence of DOM by increasing the transition probability between the singlet and the ground state. In our study, the uorescence intensity of the MW-fractionated HAs decreased with the increased molecular weight. The result may contribute to the low content of the oxygen-containing functional groups in HA fractions of higher molecular weight. [35][36][37] Similar ndings have also been reported in the MW-fractionation of DOM. 29,32,33,37 Humication index (HIX) was positively related to the MW of the fractions (except for >100 kDa HA), indicating that the extent of humication decreased with decreasing molecular weight. 33 3.2. Hetero-interaction of 4-NP with MW-fractionated HAs via an EEM uorescence spectral analysis 3.2.1. Fluorescence quenching spectra. Fluorescence quenching is a popular and powerful method due to its sensitivity, non-destructivity, and simplicity. 22 It occurs when a uorescent component interacts with a quencher, the uorescence intensity of the component weakens or disappears. Fig. 3 and S2 † showed the uorescence quenching of 4-NP by different concentrations of pristine and MW-fractionated HAs. The 4-NP presented a high uorescence intensity at Ex/Em ¼ 280/308 nm. The uorescence intensity of the peak was gradually reduced with increasing HAs concentrations due to the suppression of energy emission. 38 At an identical HA concentration, the reduction of the uorescence intensity was correlated with the molecular weight of the HAs, indicating a strong uorescence quenching effect of the high molecular weight of MWfractionated HAs on 4-NP (Fig. S3 †).
Generally, uorescence quenching includes dynamic and static quenching. In order to conrm the quenching mechanism, the Stern-Volmer model was utilized and the exponential curves were presented in Fig. S4. † It can be seen that all the titration data were well tted with the Stern-Volmer equation (R 2 ¼ 0.995-0.999), and the highly linear plot indicated a sole dynamic or static quenching process was responsible for the uorescence quenching of 4-NP by the HAs. 38,39 The quenching constant of K q for pristine and MW-fractionated HAs were signicantly larger than the maximum scatter collision quenching constant reported for dynamic quenching (2.0 Â 10 10 L mol À1 s À1 ), 27 indicating that static quenching dominated the quenching process and a ground-state complex formed Fig. 2 The fluorescence EEM spectra of the 5 mg C L À1 pristine-and MW-fractionated HAs. Fig. 3 The fluorescence EEM spectra of 4-NP in the absence and presence of 5 mg C L À1 pristine-and MW-fractionated HAs; T ¼ 298 K; pH ¼ 6.0, 1 mol L À1 NaNO 3 .
between HAs and 4-NP. For an interaction involving static quenching mechanism, the site-binding equation was applied to determine the K and n. As listed in Table 1, the log K HA of the MW-fractionated HAs decreased as the molecular weight of the MW-fractionated HAs decreased, displaying a clear MWdependent interaction heterogeneity. The results indicated the higher interaction affinities for the high molecular weight MWfractionated HA. [12][13][14][15]40 The values of n were approximately 1, suggesting a single binding site in 4-NP for MW-fractionated HAs, which was similar to the previous studies of disinfection byproducts-human serum albumin binary systems 41 and antibiotics-HA. 20 3.2.2. Thermodynamic parameters. In order to interpret the possible interaction forces between 4-NP and HAs, the interaction of HAs to 4-NP at different temperatures were performed and the corresponding thermodynamic parameters, including DH, DS, and DG were calculated. As listed in Table 1, the K and K HA values MW-fractionated HAs also exhibited similar trends, which were decreased with the molecular weight of HAs decreasing. K and K HA increased with increasing temperature for all HAs, suggesting that the interaction properties of 4-NP for MW-fractionated HAs became stronger with increasing temperature. As we know, dynamic quenching is enhanced by increasing temperature, whereas decreasing temperature most likely causes higher static quenching constants. The temperature-dependent results further demonstrated that the interactions were static quenching.
The negative DG values indicated that the interaction processes were thermodynamically spontaneous ( Table 1). The highest negative DG value in the 4-NP > 100 kDa HA system was observed at the same temperature, manifesting that >100 kDa HA possessed the largest interaction potential for 4-NP. 20 The negative DH values implied that the interaction processes were exothermic, and the positive values of DS suggested the disorder of the reaction systems increased aer 4-NP interaction with MW-fractionated HAs. Previous studies 41,42 reported that the positive DH and DS values were frequently considered as hydrophobic forces, whereas negative DH and DS values occurred for van der Waals forces and hydrogen bonding; besides, if DH < 0 and DS > 0, the interaction process was mainly driven by electrostatic forces. Therefore, the positive values of DH and DS observed in this study indicated that hydrophobic forces were responsible for the interactions between MWfractionated HAs and 4-NP under the experimental condition (pH 6, 1 mM NaNO 3 ).

Interactions between 4-NP and MW-fractionated HAs at different pH values and ionic strengths.
Ionizable organic pollutants binding to HA exhibits a strong pHdependence, reecting the speciation of both the sorbent and sorbate. 41,43 The binding parameters at various pH values were listed in Table S3. † The uorescence emission spectra of the 4-NP-HAs systems at pH 3.0 and pH 11.0 were provided in Fig. S5 and S6. † The relationship of K HA vs. pH was presented in Fig. 4a. At pH 3.0, the carboxyl groups of HAs were protonated, resulting Table 1 Binding parameters and thermodynamic parameters of 4-NP-HAs systems based on the fluorescence titration experiments (pH 6.0, 1 mmol L À1 NaNO 3 )

HAs
T (K)  in a reduction of polarity. At the same time, protonation of HAs facilitated intra-and intermolecular interactions, leading to the formation of additional hydrophobic cavities in which more hydrophobic compounds were adsorbed. 44 With the increasing pH, the molecular would extend and uncoil due to the deprotonation of phenolic and carboxyl groups. Consequently, MWfractionated HAs become less hydrophobic and more polar, which was unfavorable for binding of 4-NP. 45 Furthermore, with further increasing to pH 11.0, the 4-NP mainly existed as foremost ionic forms when pH was higher than pK a (10.7). As the electronegativity of HAs also enhanced with the elevating pH, electrostatic repulsion between negative charge of HAs and 4-NP would weaken the binding strength. 28 Generally, a signicant reduction of K HA for low molecular weight MW-fractionated HAs was observed with increasing pH (Fig. S7a †). The different extent of decrease among HA fractions was probably attributed to their respective compositional and structural characteristics. 46 As shown in Table S2, † low molecular weight MW-fractionated HAs were more enriched in polar groups and have much higher polarity compare with other fractions. Consequently, <3 kDa HA could be easily bound via molecular interactions at lower pH, resulting in more sensitivity to pH. 46 In addition, IS would affect the interactions by destroying the HA structure and compressing the double electric layers on molecule surface. 20,47 The uorescence emission spectra of the 4-NP-HAs systems at 10 mM and 100 mM NaNO 3 were provided in Fig. S8 and S9. † Fig. 4b showed the binding constants of MWfractionated HAs dramatically decreased with NaNO 3 concentration from 1 mmol L À1 to 100 mmol L À1 at neutral pH solution. All MW-fractionated HAs exhibited a similar trend. The high concentration ions compressed the thickness of the electric double layer of HA colloids effectively, inducing HA aggregation and even precipitation. 45,48 This tightly packed molecular shape of HA would reduce the capability of 4-NP to interact with HA. 49 Furthermore, the decrease of the K HA in high molecular weight MW-fractionated HAs was smaller than that in low molecular weight MW-fractionated HAs (Fig. S7b †). The reduction of K HA was mainly deemed to the change of HAs molecular size, which was caused by the associations between cations and acidic groups. Thus, the contents of negatively charged acidic functional groups of HAs were probably responsible for the different results among MW-fractionated HAs, which was consistent with the experimental results for hydrophobic fractions and different origins of DOM. 49,50

UV-vis, FT-IR, 1 H NMR spectra of 4-NP-HAs complexations
The UV-vis absorption spectra of 4-NP in the absence and presence of MW-fractionated HAs at pH 6 and 1 mM NaNO 3 were measured and presented in Fig. 5a. With addition of equivalent MW-fractionated HAs, the absorption intensities and shapes of 4-NP changed. As the molecular weight of fractionated HAs decreased, a slight blue shi was observed for the absorption peak at around 275 nm. 20 The apparent variation indicated that steady 4-NP-HAs complexes were formed and the molecular weight of HA had a signicant effect on the micro-environmental changes of 4-NP. Besides, the distinct changes further conrmed that static quenching occurs in the interactions. This was because the dynamic quenching hardly affected absorption spectra of quenching matters.
To explore the changes of MW-fractionated HAs functional groups complexing with 4-NP, the FT-IR spectra of 4-NP exposed to HA at pH 6 and 1 mM NaNO 3 were collected and shown in Fig. 5b. Compared with the MW-fractionated HAs spectra, the signicantly increased absorption intensity at 1380, 1240, and 1124 cm À1 was attributed to C-O stretching vibration, phenolic O-H, and C-H bending vibration of 4-NP, respectively. 51 The peaks of the H-bonded OH vibration moved slightly to the low wavenumber, and the peaks were broadened when exposed to HAs, implying H-bonding involving the 4-NP in HA. 20 The C]C peak of MW-fractionated HAs aer binding shied from 1635 to 1596 cm À1 , which could be attributed to the p-p interactions between the benzene rings of 4-NP and MW-fractionated HAs. 39,52 These features of the FTIR spectra proved that the 4-NP was not mechanically mixed with the HAs but bound to the MW-fractionated HAs.
Nuclear magnetic resonance (NMR) was applied to provide molecular-level information involved in the reactions between 4-NP and HAs. As shown in Fig. 5c, the chemical shis above 6.2 ppm were attributed to the 4-NP protons of the aromatic region, while the signals resonating between 1 and 4.7 ppm corresponded to protons in the aliphatic regions. Due to the lower concentration of MW-fractionated HAs, almost no proton NMR signal was observed (Fig. S10 †). Upon addition of MWfractionated HAs, the proton signals of 4-NP were quenched and broadened, suggesting a more rapid relaxation rate due to dipolar interactions arising from restricted mobility of 4-NP molecules. 20,53,54 Simultaneously, the chemical shis of aromatic proton signals moved upeld due to the increased electron shielding around proton nuclei. 23 Though similar trends were observed in all 4-NP-HAs systems, the extent of signal changes (H3, H4 and H2, H5) was larger for high molecular weight MWfractionated HAs than that of low molecular weight MWfractionated HAs (Fig. S11 †). These observations might be attributed to the establishment of p-p interactions between 4-NP and the aromatic parts of HAs, where the center of magnetic anisotropy was shied out of the 4-NP aromatic plane toward the center of newly formed p-p complexes. 20,53 The strong p-p interactions would be formed between 4-NP and high molecular weight MW-fractionated HAs, leading to increasing the electron shielding of proton nuclei. In addition, the nuclei of 4-NP entrapped in higher hydrophobic cavities of >100 kDa HA might induce an improved magnetic shielding, causing a larger upeld shi. 55,56 In previous studies, the hydrogen bonding interaction was also used to explain the upeld shi between HA and aromatic organic pollutants. 20,57 However, the chemical shi variation was smaller for more hydrophilic and nonpolar MWfractionated HAs. Therefore, the interaction between 4-NP and HAs should not be primarily related to H-bonding at high pH. 53 Overall, p-p interactions between aromatic rings of 4-NP and MW-fractionated HAs and hydrophobic interactions played important roles in the interaction process.

Relationships of the physicochemical properties with interaction properties
The varied interaction properties of MW-fractionated HAs may be related to their distinct chemical compositions and structures. As shown in Fig. 6, the log K HA values are strongly positively correlated with SUVA 254 (R 2 > 0.872), implying that the aromatic structures of HAs have critical importance in the interaction process of 4-NP with MW-fractionated HAs. The results were consistent with the studies of HOCs interaction with MW-fractionated HAs. 13,14,58,59 HIX was also positively correlated with the log K HA . Higher values of HIX suggested higher degrees of aromaticity, lower rates of mineralization, and lower contents of oxygen-containing functional groups for DOM. 40 The importance of humication degree was highlighted in the interaction process between HOCs and DOM fractions. 14,15 Additionally, (O + N)/C atomic ratios, as an index of the polarity of HA, negatively correlated with log K HA values. Similarly, Jin et al. 58 observed that HA fractions with higher polarity had lower interaction properties for phenanthrene than other HA fractions. It has been proposed that the hydrophilic moieties of HA would reduce the accessibility of HOCs to sorption domains and compete for sorption sites, thus decreasing their interactions. 60 Taken together, the superior interaction property for the high molecular weight MW-fractionated HAs resulted from its higher aromaticity, humication degree, and lower polarity.
Principal component analysis (PCA) is a multivariate statistical technique that can be used to simplify the selected parameters and convert the data into graphical form. To further understand the hetero-interaction of 4-NP with HA, PCA was performed by using the selected parameters of the HA samples (e.g., C, H, O, N, MW, Mn, HIX, SUVA 254 , O/C, (N + O)/C, BIX (biological index), FI (uorescence index), K HA , K, n) that had relationships with formation of 4-NP-HAs complex. As seen in Fig. 7a, the percentage of variance of principal component 1 (PC1) and principal component 2 (PC2) was approximately 78.4% and 10.3% in the HA samples, respectively. The cumulative percentage of variance of PC1 and PC2 is 88.7%, indicating the interaction behavior mainly considered the two components of PC1 and PC2. The factors associated with aromaticity and MW were loaded mainly by PC1 and were positively related to PC1. The high statistically signicant correlation among MW, Mn, HIX, SUVA 254 , K HA suggest that these factors are similar. In addition, the loadings graph also exhibited that K HA has a strong relationship with SUVA 254 , MW and HIX, conrming that aromatic C in MW-fractionated HAs may signicantly take part in the interactions. These results are vital for elucidating the formation of 4-NP-HAs in the future. Furthermore, in the scores graph (Fig. 7b), the distribution of different types of HA was obvious distinct, suggesting a clear interaction heterogeneity among MW-fractionated HAs sample, and proving that the critical differences in structural characteristics resulted in a difference in the 4-NP-HA complex.

Environmental implication
4-NP has been broadly distributed in aquatic environments. Thus, highly hydrophobic NPs 4-NP has inevitably interacted with ubiquitous HA in the environments. In this study, the HA samples used were environmentally related concentrations, which indicated the feasibility of extrapolating these results to natural ecosystems. Additionally, previous work about 4-NP-HA interaction mainly focused on the bulk HA samples, ignoring the effects of the heterogeneities within the bulk HA on the interaction behaviors. 10 This work highlighted MW-dependent interaction heterogeneities of HA, which suggested that heterogeneity in MW distribution should be taken into consideration when exploring the 4-NP-HA interactions and assessing the fate and transport of 4-NP in aquatic environments.
4-NP could interact with MW-fractionated HAs to form a relative stable complex spontaneously. Generally, higher molecular weight MW-fractionated HAs have stronger sorption ability to 4-NP than lower higher molecular weight MWfractionated HAs. Via a correlation analysis and PCA, the results indicated that aromaticity and MW played vital roles in the interaction process. Higher MW of HA accompanied with higher aromatic moieties had prone to interaction with 4-NP, which might signicantly inuence the biodegradation of 4-NP. As Lin et al. 12 pointed out, the DOM concentration and structure, organism species, exposure regime. As well as water conditions could inuence the biodegradation of pollutants. Thus, the biodegradation of MW-fractionated HAs-associated 4-NP are needed to be precisely investigated in further researches. Moreover, the formation of 4-NP-HAs complex was mainly induced by hydrophobic interactions and p-p interactions, which could be interfered by different pHs and ISs. These results herein were helpful for understanding the effects of MW distribution of HA on the interaction with 4-NP under environmentally relevant conditions.

Conclusions
The interaction of the MW-dependent fractionated HAs with 4-NP were explored by EEM, UV-visible spectroscopy, FT-IR, and 1 H NMR analysis. The quenching processes of all samples were static, and the binding affinities calculated from the Stern-Volmer and site-binding equations reduced with the MW decreased, exhibiting a clear MW-dependent binding heterogeneity in aquatic environments. The binding constants for 4-NP-HAs system were suppressed as the ionic strength decreased and pH increased. The binding reactions occurred spontaneously, and the interactions were mainly driven by hydrophobic interaction. Additionally, p-p interactions between aromatic rings of 4-NP and MW-fractionated HAs might play crucial roles in forming the 4-NP-HAs complex, as indicated by FT-IR and 1 H NMR. This study highlighted the importance of MW in HA interaction with 4-NP, which can inuence the fate and behavior of 4-NP in aquatic environments. Besides heterogeneity, the regional and temporal variation in different sources of HA also has important impacts on the interaction, which should be further assessed for predicting the behavior of 4-NP in natural environments.